Characterization of ion channels in human preadipocytes.
Ion channels participate in regulation of cell proliferation. However, though preadipocyte (the progenitor of fat cell) is a type of highly proliferating cells, ion channel expression and their role in proliferation is not understood in human preadipocytes. The present study was designed to characterize ion channels using whole-cell patch clamp technique, RT-PCR, and Western blotting. It was found that a 4-aminopyridine- (4-AP) sensitive transient outward K(+) current (I(to)) was present in a small population of (32.0%) cells, and an outward "noisy" big conductance Ca(2+)-activated K(+) current (I(KCa)) was present in most (92.7%) preadipocytes. The noisy current was inhibited by the big conductance I(KCa) channel blocker paxilline (1 microM), and enhanced by the Ca(2+) ionophore A23187 (5 microM) and the big conductance I(KCa) channel activator NS1619 (10 microM). RT-PCR and Western blot revealed the molecular identities (i.e., KCa1.1 and Kv4.2) of the functional ionic currents I(KCa) and I(to). Blockade of I(KCa) or I(to) with paxilline or 4-AP reduced preadipocyte proliferation, and similar results were obtained with specific siRNAs targeting to KCa1.1 and Kv4.2. Flow cytometric analysis showed ion channel blockade or knockdown of KCa1.1 or Kv4.2 with specific siRNA increased the cell number of G0/G1 phase. The present study demonstrates for the first time that two types of functional ion channel currents, I(to) and big conductance I(KCa), are present in human preadipocytes and that these two types of ion channels participate in regulating proliferation of human preadipocytes.
Hu
Hao
H
Department of Medicine, Research Centre of Heart, Brain, Healthy Aging, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong SAR, China.
He
Mu-Lan
ML
Tao
Rong
R
Sun
Hai-Ying
HY
Hu
Rui
R
Zang
Wei-Jin
WJ
Yuan
Bing-Xiang
BX
Lau
Chu-Pak
CP
Tse
Hung-Fat
HF
Li
Gui-Rong
GR
D015761:4-Aminopyridine / Q000494:pharmacology; D017667:Adipocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D050156:Adipogenesis / Q000187:drug effects; D015153:Blotting, Western; D002453:Cell Cycle / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D015536:Down-Regulation / Q000187:drug effects; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D015640:Ion Channel Gating / Q000187:drug effects; D007473:Ion Channels / Q000378:metabolism*; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits / Q000235:genetics / Q000378:metabolism; D049990:Membrane Transport Modulators / Q000494:pharmacology; D024681:Potassium Channels, Calcium-Activated / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051672:Shal Potassium Channels / Q000235:genetics / Q000378:metabolism
D007211:Indoles; D007473:Ion Channels; C494841:KCNMA1 protein, human; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D049990:Membrane Transport Modulators; D024681:Potassium Channels, Calcium-Activated; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D051672:Shal Potassium Channels; C048220:paxilline; D015761:4-Aminopyridine